Spatially encoded biological assays

ABSTRACT

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/430,015, filed Jun. 3, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/414,213, filed on May 16, 2019, which is acontinuation of U.S. patent application Ser. No. 16/402,098 filed May 2,2019, which is a continuation of U.S. patent application Ser. No.16/276,235 filed Feb. 14, 2019, which is a continuation application ofU.S. patent application Ser. No. 15/187,661 filed Jun. 20, 2016 (nowU.S. Pat. No. 10,308,892), which is a continuation of Ser. No.13/080,616 filed Apr. 5, 2011 (now U.S. Pat. No. 9,371,598), whichclaims the benefit of U.S. Provisional Patent Application No.61/321,124, filed Apr. 5, 2010, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to assays of biological molecules, and moreparticularly to assays for determining spatial distributions of a largenumber of biological molecules in a solid sample simultaneously.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Comprehensive gene expression analysis and protein analysis have beenuseful tools in understanding mechanisms of biology. Use of these toolshas allowed the identification of genes and proteins involved indevelopment and in various diseases such as cancer and autoimmunedisease. Conventional methods such as in situ hybridization and othermultiplexed detection of different transcripts have revealed spatialpatterns of gene expression and have helped shed light on the molecularbasis of development and disease. Other technologies that have enabledthe quantitative analysis of many RNA sequences per sample includemicroarrays (see Shi, et al., Nature Biotechnology, 24(9):1151-61(2006); and Slonim and Yanai, Plos Computational Biology, 5(10):e1000543(2009)); serial analysis of gene expression (SAGE) (see Velculescu, etal, Science, 270(5235):484-87 (1995)), high-throughput implementationsof qPCR (see Spurgeon, et al., Plos ONE, 3(2):e1662 (2008)) and in situPCR (see Nuovo, Genome Res., 4:151-67 (1995)). As useful as thesemethods are, however, they do not enable simultaneous measurement of theexpression of many genes or the presence and/or activity of multipleproteins at many spatial locations in a sample. Laser capturemicrodissection has permitted the analysis of many genes at a smallnumber of locations, but it is very expensive, laborious, and does notscale well. Certain PCR assays in a 2D format preserve spatialinformation (see Armani, et al., Lab on a Chip, 9(24): 3526-34 (2009)),but these methods have low spatial resolution because they rely onphysical transference of tissue into wells, which also prevents randomaccess to tissue samples and high levels of multiplexing.

At present, no practical method exists to analyze at high resolution thespatial expression patterns of large numbers of genes, proteins, orother biologically active molecules simultaneously. There is thus a needfor reproducible, high-resolution spatial maps of biological moleculesin tissues. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The invention encompasses assay systems that provide high-resolutionspatial maps of biological activity in tissues. The assay systemcomprises an assay capable of high levels of multiplexing where encodedprobes are provided to a biological sample in defined spatial patterns;instrumentation capable of controlled delivery of reagents according tothe spatial patterns; and a decoding scheme providing a readout that isdigital in nature. In short, the present invention provides the abilityto look at many biological targets in many locations, providing theresolution of in situ hybridization with the highly-parallel dataanalysis of sequencing.

Thus, in some embodiments, the invention provides an assay system todetermine spatial patterns of abundance or activity or both of multiplebiological targets at multiple sites in a sample, where the assay systemperforms the following steps: providing a sample affixed to a support;delivering encoded probes for the multiple biological targets to themultiple sites in the sample in a known spatial pattern, where eachencoded probe comprises a probe region that may interact with thebiological targets and a coding tag that identifies a location of thesite to which the encoded probe was delivered; allowing the encodedprobes to interact with the biological targets; separating encodedprobes that interact with the biological targets from encoded probesthat do not interact with the biological targets; determining all or aportion of a sequence of the encoded probes, and associating theabundance or activity or both of the multiple biological targets to thelocations of the sites in the sample.

In particular aspects of the invention the biological targets comprisenucleic acids and the encoded probes are oligonucleotides, and in someaspects, there are two encoded probes for each of the multiple nucleicacid targets. In some aspects, the multiple biological targets compriseproteins, the probe regions of the encoding probes are proteins and thecoding tags comprise oligonucleotides. In some aspects the multiplebiological targets comprise enzymes. In some aspects the probe regionsof the encoded probes comprise antibodies, aptamers or small molecules.

Some aspects of the assay system further comprise an amplification stepbetween the separating step and the determining step. In some aspects,the determining step is performed by nucleic acid sequencing, and inpreferred aspects, the sequencing is high-throughput digital nucleicacid sequencing.

In some aspects of the invention, the product of the multiple biologicaltargets being assayed and the multiple sites in the sample is greaterthan 20, in some aspects product of the multiple biological targetsbeing assayed and the multiple sites in the sample is greater than 50,in some aspects the product of the multiple biological targets beingassayed and the multiple sites in the sample is greater than 75, 100,150, 500, 750, 1,000, 5,000, 10,000, 25,000, 50,000, 100,000, 500,000,or 1,000,000 or more. In other aspects, the sequence of at least fiftythousand encoding probes are determined in parallel, in other aspectsthe sequence of at least one hundred thousand encoding probes aredetermined in parallel, in some aspects the sequence of at least fivehundred thousand encoding probes are determined in parallel, and in someaspects the sequence of at least one million, ten million, one hundredmillion, one billion, ten billion, one hundred billion or more encodingprobes are determined in parallel.

In some aspects, the known spatial pattern is determined by histologicalfeatures of the sample. Also in some aspects, software programmedhardware performs at least two steps of the delivering step, theseparation step, the determining step and the associating step.

In some aspects, the probe regions of the encoded probes are proteinsand the separating step is accomplished by encoded probes that interactwith the biological targets being captured by an affinity capture agent.In some aspects the probe regions of the encoding probes are nucleicacids and the separating step is accomplished by a washing of thesample.

In other embodiments there is provided an assay system to determinespatial patterns of abundance or activity or both of multiple nucleicacid targets at multiple sites in a sample, where the assay systemperforms the following steps: providing a sample affixed to a support;delivering oligonucleotide probes for multiple nucleic acid targets tothe multiple sites in the sample in a known spatial pattern; allowingthe oligonucleotide probes to hybridize with the nucleic acid targets;washing unhybridized encoded oligonucleotide probes from the sample;delivering one or more encoding agents to locations of the multiplesites in the sample according to a known spatial pattern, where thecombination of encoding agents delivered to each site is different;coupling the encoding agents and the oligonucleotide probes to formencoded probes; determining all or a portion of a sequence of theencoded probes using high-throughput sequencing, and associating theabundance or activity or both of multiple biological targets to thelocations of multiple sites in the sample.

Other embodiments of the invention provide an assay system to determinespatial patterns of abundance or activity or both of multiple proteintargets at multiple sites in a sample, where the assay system performsthe following steps: providing a sample affixed to a support; deliveringencoded probes for the multiple protein targets to the multiple sites inthe sample in a known spatial pattern, where each encoded probecomprises a protein probe region that may interact with the proteintargets and a coding tag that identifies a location of the site to whichthe encoded probe was delivered and the protein probe region of theencoding probe of which the coding tag is part; allowing the encodedprobes to interact with the protein targets; separating encoded probesthat interact with the protein targets from encoded probes that do notinteract with the protein targets; determining all or a portion of asequence of the encoded probes by high throughput sequencing, andassociating the abundance or activity or both of the multiple proteintargets to the locations of the multiple sites in the sample.

Other embodiments provide an assay system to determine spatial patternsof abundance or activity or both of multiple biological targets atmultiple sites in a sample, where the assay system performs thefollowing steps: providing a sample affixed to a support; deliveringencoded probes for the multiple biological targets to the multiple sitesin the sample in a known spatial pattern, where each encoded probecomprises a probe region that may interact with the biological targetsand a coding tag that identifies a location of the site to which theencoded probe was delivered and identifies the biological target;allowing the encoded probes to interact with the biological targets;determining all or a portion of a sequence of the encoded probes, andassociating the abundance or activity or both of the multiple biologicaltargets to the locations of the sites in the sample.

The assay system of the invention can utilize various detectionmechanisms, based on the molecules to be detected and the reagentsneeded for such detection system. Exemplary methods that can be usedwith the assay systems of the invention are described in more detailbelow.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a simplified overview of the assay system of the presentinvention.

FIG. 2 provides a simplified overview of one embodiment of the assaysystem of the present invention for detecting nucleic acids.

FIG. 3 is a representational depiction of one embodiment of the assayoverviewed in FIG. 2.

FIG. 4A-C illustrates a general mechanism for one embodiment of acombinatorial encoding scheme of the assay systems of the invention.FIG. 4A shows two target-specific/encoding oligonucleotide constructsspecifically bound to a target nucleic acid of interest in a sample.FIG. 4B shows a scheme for delivering twenty different coding tags, a1through a10 and b1 through b10, to a sample to form a 10×10 coding taggrid. FIG. 4C shows a tissue section sample to which the coding tags aredelivered, forming the coding tag grid in the sample.

FIG. 5 provides a simplified, specific example of the embodiment of acombinatorial encoding scheme shown in FIG. 4.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “antibody” as used herein is intended to refer to an entireimmunoglobulin or antibody or any functional fragment of animmunoglobulin molecule which is capable of specific binding to anantigen (antibodies and antigens are “binding partners” as definedherein). “Antibody” as used herein is meant to include the entireantibody as well as any antibody fragments capable of binding theantigen or antigenic fragment of interest. Examples of such peptidesinclude complete antibody molecules, antibody fragments, such as Fab,F(ab′)2, CDRS, VL, VH, and any other portion of an antibody which iscapable of specifically binding to an antigen. Antibodies for assays ofthe invention are immunoreactive or immunospecific for, and thereforespecifically and selectively bind to, proteins either detected (i.e.,biological targets) or used for detection (i.e., probes) in the assaysof the invention.

The term “binding agent” as used herein refers to any agent thatspecifically binds to a biological molecule of interest

“Complementary” or “substantially complementary” refers to thehybridization or base pairing or the formation of a duplex betweennucleotides or nucleic acids, such as, for instance, between the twostrands of a double-stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single-stranded nucleic acid.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single-stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the other strand, usually at least about 90%to about 95%, and even about 98% to about 100%).

“Hybridization” refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-strandedpolynucleotide, The resulting (usually) double-stranded polynucleotideis a “hybrid” or “duplex,” “Hybridization conditions” will typicallyinclude salt concentrations of approximately less than 1M, often lessthan about 500 mM and may be less than about 200 mM. A “hybridizationbuffer” is a buffered salt solution such as 5% SSPE, or other suchbuffers known in the art. Hybridization temperatures can be as low as 5°C., but are typically greater than 22° C., and more typically greaterthan about 30° C., and typically in excess of 37° C. Hybridizations areoften performed under stringent conditions, i.e., conditions under whicha primer will hybridize to its target subsequence but will not hybridizeto the other, non-complementary sequences. Stringent conditions aresequence-dependent and are different in different circumstances. Forexample, longer fragments may require higher hybridization temperaturesfor specific hybridization than short fragments. As other factors mayaffect the stringency of hybridization, including base composition andlength of the complementary strands, presence of organic solvents, andthe extent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one parameter alone.Generally stringent conditions are selected to be about 5° C. lower thanthe T_(m) for the specific sequence at a defined ionic strength and pH.Exemplary stringent conditions include a salt concentration of at least0.01 M to no more than 1M sodium ion concentration (or other salt) at apH of about 7.0 to about 8.3 and a temperature of at least 25° C. Forexample, conditions of 5×SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mMEDTA at pH 7.4) and a temperature of approximately 30° C. are suitablefor allele-specific hybridizations, though a suitable temperaturedepends on the length and/or GC content of the region hybridized.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g., oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon terminal nucleotide of one oligonucleotide with a 3′ carbon ofanother nucleotide.

“Nucleic acid”, “oligonucleotide”, “oligo” or grammatical equivalentsused herein refers generally to at least two nucleotides covalentlylinked together. A nucleic acid generally will contain phosphodiesterbonds, although in some cases nucleic acid analogs may be included thathave alternative backbones such as phosphoramidite, phosphorodithioate,or methylphophoroamidite linkages; or peptide nucleic acid backbones andlinkages. Other analog nucleic acids include those with bicyclicstructures including locked nucleic acids, positive backbones, non-ionicbackbones and non-ribose backbones. Modifications of theribose-phosphate backbone may be done to increase the stability of themolecules; for example, PNA:DNA hybrids can exhibit higher stability insome environments.

“Primer” means an oligonucleotide, either natural or synthetic, that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.The sequence of nucleotides added during the extension process isdetermined by the sequence of the template polynucleotide. Primersusually are extended by a DNA polymerase.

The term “SNP” or “single nucleotide polymorphism” refers to a geneticvariation between individuals; e.g., a single nitrogenous base positionin the DNA of organisms that is variable. SNPs are found across thegenome; much of the genetic variation between individuals is due tovariation at SNP loci, and often this genetic variation results inphenotypic variation between individuals. SNPs for use in the presentinvention and their respective alleles may be derived from any number ofsources, such as public databases (U.C. Santa Cruz Human Genome BrowserGateway (http://genome.ucsc.edu/cgi-bin/hgGateway) or the NCBI dbSNPwebsite (http://www.ncbi.nlm.nih.gov/SNP/), or may be experimentallydetermined as described in U.S. Pat. No. 6,969,589; and US Pub. No.2006/0188875 entitled “Human Genomic Polymorphisms.” Although the use ofSNPs is described in some of the embodiments presented herein, it willbe understood that other biallelic or multi-allelic genetic markers mayalso be used. A biallelic genetic marker is one that has two polymorphicforms, or alleles. As mentioned above, for a biallelic genetic markerthat is associated with a trait, the allele that is more abundant in thegenetic composition of a case group as compared to a control group istermed the “associated allele,” and the other allele may be referred toas the “unassociated allele.” Thus, for each biallelic polymorphism thatis associated with a given trait (e.g., a disease or drug response),there is a corresponding associated allele. Other biallelicpolymorphisms that may be used with the methods presented hereininclude, but are not limited to multinucleotide changes, insertions,deletions, and translocations. It will be further appreciated thatreferences to DNA herein may include genomic DNA, mitochondrial DNA,episomal DNA, and/or derivatives of DNA such as amplicons, RNAtranscripts, cDNA, DNA analogs, etc. The polymorphic loci that arescreened in an association study may be in a diploid or a haploid stateand, ideally, would be from sites across the genome.

The term “selectively binds”, “selective binding” and the like as usedherein, when referring to a binding partner (e.g. protein, nucleic acid,antibody or other affinity capture agent, etc.), refers to a bindingreaction of two or more binding partners with high affinity and/orcomplementarity to ensure selective hybridization under designated assayconditions. Typically, specific binding will be at least three times thestandard deviation of the background signal. Thus, under designatedconditions the binding partner binds to its particular “target” moleculeand does not bind in a significant amount to other molecules present inthe sample.

“Sequencing”, “sequence determination” and the like means determinationof information relating to the nucleotide base sequence of a nucleicacid. Such information may include the identification or determinationof partial as well as full sequence information of the nucleic acid.Sequence information may be determined “with varying degrees ofstatistical reliability or confidence. In one aspect, the term includesthe determination of the identity and ordering of a plurality ofcontiguous nucleotides in a nucleic acid, “High throughput digitalsequencing” or “next generation sequencing” means sequence determinationusing methods that determine many (typically thousands to billions) ofnucleic acid sequences in an intrinsically parallel manner, i.e. whereDNA templates are prepared for sequencing not one at a time, but in abulk process, and where many sequences are read out preferably inparallel, or alternatively using an ultra-high throughput serial processthat itself may be parallelized. Such methods include but are notlimited to pyrosequencing (for example, as commercialized by 454 LifeSciences, Inc., Branford, Conn.); sequencing by ligation (for example,as commercialized in the SOLiD™ technology, Life Technology, Inc.,Carlsbad, Calif.); sequencing by synthesis using modified nucleotides(such as commercialized in TruSee™ and HiSeg™ technology by Illumina,Inc., San Diego, Calif., HeliScope™ by Helicos Biosciences Corporation,Cambridge, Mass., and PacBio RS by Pacific Biosciences of California,Inc., Menlo Park, Calif.), sequencing by ion detection technologies (IonTorrent, Inc., South San Francisco, Calif.); sequencing of DNA nanoballs(Complete Genomics, Inc., Mountain View, Calif.); nanopore-basedsequencing technologies (for example, as developed by Oxford NanoporeTechnologies, LTD, Oxford, UK), and like highly parallelized sequencingmethods.

The term “T_(m)” is used in reference to the “melting temperature.” Themelting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. Several equations for calculating the T_(m) of nucleicacids are well known in the art. As indicated by standard references, asimple estimate of the T_(m) value may be calculated by the equation,T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at1M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)). Other references(e.g., Allawi and SantaLucia, Jr., Biochemistry, 36:10581-94 (1997))include alternative methods of computation which take structural andenvironmental, as well as sequence characteristics into account for thecalculation of T_(m).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols.I-IV) (1999); Weiner, Gabriel, Stephens, Eds., Genetic Variation: ALaboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: ALaboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: AMolecular Cloning Manual (2003); Mount, Bioinformatics: Sequence andGenome Analysis (2004); Sambrook and Russell, Condensed Protocols fromMolecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell,Molecular Cloning: A Laboratory Manual (2002) (all from Cold SpringHarbor Laboratory Press); Stryer, Biochemistry (4th Ed.) (1995) W.H,Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” (2002) IRL Press, London; Nelson and Cox, Lehninger,Principles of Biochemistry (2000) 3^(rd) Ed., W. H. Freeman Pub., NewYork, N.Y; and Berg, et al., Biochemistry (2002) 5^(th) Ed., W. H.Freeman Pub., New York, N.Y., all of which are herein incorporated intheir entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a nucleic acid”refers to one or more nucleic acids, and reference to “the assay”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention,

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The Invention in General

The assay systems of the invention provide spatially-encoded,multiplexed assays comprising 1) an assay capable of high levels ofmultiplexing with an efficient spatial encoding scheme; 2)instrumentation capable of delivering reagents according to a spatialpattern; and 3) decoding determined by a readout that is digital innature. The assay systems of the invention detect the presence orabsence and relative amount of a biological target or biologicalactivity indicative of a biological target, as well as the location ofthe biological target or activity in a biological sample, e.g., a tissuesection or other biological structure disposed upon a support such as amicroscope slide or culture dish.

The assay system further provides instrumentation with an ability todeliver reagents in a spatially-defined pattern. This instrumentation,together “with software, reagents and protocols, provides a keycomponent of the highly innovative assay system of the invention,allowing for measurement of numerous biological targets or activities ina meaningful spatial environment, including gene expression and peptidelocalization, An encoding scheme used in these assay systems allows oneto determine the location of biological targets or activity (or lackthereof) in the biological samples after the products of the multiplexedassay are removed from the biological sample and pooled for analysis.Decoding of the encoding scheme can be performed by, e.g.,next-generation sequencing, which easily provides millions to trillionsof data points at low cost. The assay results such as the amount oractivity of biological targets can then be mapped back to specificlocation in the biological sample. The assay systems open a newanalytical window into the complex spatial patterns of cellular functionand regulation in biological samples.

A simplified overview of the assay system 100 of the present inventionis provided at FIG. 1, At step 110, a biological sample affixed to asupport is provided. The biological sample contains biological targetsof interest. Biological targets can include any molecule of interest,such as nucleic acids (including, e.g, RNA transcripts, genomic DNAsequences, cDNAs, amplicons, or other nucleic acid sequences) andproteins, enzymes and the like. At step 120, encoded probes aredelivered to the biological sample according to a known spatial pattern.Encoded probes comprise probes, which can interact “with biologicaltargets of interest, and coding tags, which identify the positions inthe sample of the biological targets being assayed, and thus can be usedto link assay results back to locations in the sample. Coding tags inmost embodiments are oligonucleotides. However, coding tags may also bemass tags, fluorescent labels, or other moieties.

In some embodiments, the probe and coding tag portions of the encodedprobe are pre-coupled before being delivered to the biological sample,For example, in the case where the encoded probes are oligonucleotides,both the probe and coding tag sequence can be synthesized as a singleoligonucleotide. Alternatively, the probe and coding tag portions of theencoding probes can be synthesized or obtained separately and combinedbefore delivery to the biological sample (e.g., two separateoligonucleotides can be synthesized and coupled by, e.g., ligation; oran antibody and an oligonucleotide can be prepared separately andconjugated before delivery to the biological sample). Also, as isdescribed in FIGS. 2-5, the probes and the coding tags (in encodingoligonucleotides) are synthesized separately, and are delivered to thebiological sample at different steps (e.g., probes first and coding tagsthereafter, or vice versa) in the assay.

At step 130, the encoded probes are allowed to react or interact withthe biological targets, i.e., conditions are provided to allow e.g.,oligonucleotides to hybridize to nucleic acid targets, enzymes tocatalyze reactions with protein targets, antibodies to bind epitopes,etc. In the case where the biological targets are nucleic acids, theencoded probes are typically oligonucleotides and hybridize to thetarget nucleic acids. In the case that the biological targets areproteins, the encoded probes typically are aptamers, small molecules, oroligonucleotide-conjugated proteins that interact with target proteinsby binding to them or by reacting with them (that is, one of theproteins is a substrate for the other). Encoding oligonucleotides may becoupled to the probes (proteins) by conjugation, chemical orphoto-crosslinking via suitable groups and the like.

Once encoded probes interact with the biological targets, the encodedprobes that interacted with the biological targets must be separatedfrom the encoded probes that did not interact with the biologicaltargets at step 140. In the case where the biological targets arenucleic acids and the encoded probes are oligonucleotides, theseparation can be accomplished by, e.g., washing the unhybridizedencoded probes from the sample. Similarly, for other assays that arebased on affinity binding, including those using aptamer, smallmolecule, and protein probes, washing steps can be used to remove lowaffinity binders. In the case where the probe is transformed viainteraction with the target, e.g., in the case of a peptide, e.g., viacleavage by a protease or phosphorylation by a kinase, it is convenientto collect, all encoded probes—both encoded probes that interacted withthe biological targets and were transformed and encoded probes that werenot transformed. After collection or pooling, an antibody or otheraffinity capture agent can be used to capture probes that weretransformed by addition of a moiety (e.g., a phosphate group). In caseswhere probes have been transformed via cleavage, the transformed probescan be separated, e.g., by capturing the non-transformed probes via atag that is removed from the transformed probes during thetransformation (e.g., by cleavage), or by adding a new tag at the siteof cleavage.

Once the reacted (transformed) or interacted encoded probes areseparated from the unreacted or un-interacted encoded probes, thesequence of the reacted and/or interacted encoded probes is determinedat step 150 by, preferably, sequencing. The sequence of the encodedprobes allows the mapping of the assay results at step 160 back tolocations in the biological sample.

FIG. 2 provides a simplified overview of an assay system 200 of thepresent invention embodying an efficient implementation of acombinatorial coding scheme for the encoding of spatial information. Forpurposes of this overview, the probes are oligonucleotides, but asexplained elsewhere, other types of probes can also be used. In step210, a biological sample affixed to a support, e.g., a tissue sample orother biological structure, is provided. In step 220, one or moreoligonucleotide probes are delivered to the biological sample, where theoligonucleotide probes are capable of hybridizing with biologicaltargets in the biological sample. In step 230, the oligonucleotideprobes are allowed to interact with (hybridize to) the nucleic acidtargets; that is, appropriate conditions are provided whereoligonucleotide probes can hybridize to the target nucleic acids.

In step 240, the oligonucleotide probes that did not hybridize to targetnucleic acids are removed, and thereby separated from oligonucleotideprobes that did hybridize to target nucleic acids. In this embodiment,separation can be accomplished by, e.g., washing the sample to removeunhybridized oligonucleotide probes. Next, in step 250, encodingoligonucleotides (the encoding agents) are delivered to the biologicalsample according to a chosen spatial pattern, where the encodingoligonucleotides comprise coding tags that are used to encode thelocation of biological targets in the biological sample. Note that incontrast to the assay system of FIG. 1, here the probes and encodingagents (encoding oligonucleotides) are delivered in separate steps. Instep 260, the encoding oligonucleotides are coupled to theoligonucleotide probes to create encoded probes. In this case where theprobes are oligonucleotides, the encoding oligonucleotides may becoupled to the oligonucleotides probes by, e.g., ligation.Alternatively, the information in the encoding oligonucleotides can betransferred by using a DNA polymerase to extend a probe oligonucleotidethat acts as a primer, and thereby copy and incorporate the sequence ofthe encoding oligonucleotides.

In step 270, the sequence of the coding tags in the encoded probes aswell as the sequence or a portion of the sequence of the probe itself isdetermined, and in step 280, the target nucleic acids are mapped back tothe biological sample. In some embodiments, the abundance of sequencesreveals the relative quantity of biological targets at the location.Although this embodiment shows the individual steps in a particularorder, so as to better explain the invention, the precise order of thesteps can be varied. For example, steps 220 and 250 can be combined, sothat a mixture of the probes and encoding oligonucleotides is deliveredaccording to a chosen spatial pattern. Coupling step 260 can then becarried out immediately after the combined steps 220 and 250, orconcomitantly with them. In this case, step 240 would then occur afterstep 260. It can therefore be appreciated that the two key results ofthis series of steps, i.e., the location-specific encoding of probemolecules and the separation of probe molecules based on their abilityto interact with corresponding target molecules, can be accomplishedwith some flexibility in the implementation of the particular steps.Similarly, there is considerable flexibility in the design of the codingscheme. As described infra, the assays of the invention are particularlyamenable to combinatorial methods.

Thus, the present invention provides an ability to look at manydifferent biological targets in many locations, providing the resolutionof in situ hybridization with the highly-parallel data analysis ofsequencing. In some embodiments, the sum of the multiple biologicaltargets being assayed and the multiple sites in the biological sample isgreater than 20, in other embodiments, the sum of the multiplebiological targets being assayed and the multiple sites in thebiological sample is greater than 50, in other embodiments, the sum ofthe multiple biological targets being assayed and the multiple sites inthe biological sample is greater than 100, greater than 500, 1,000,10,000, 25,000, 100,000, 500,000, 1,000,000. It will be appreciatedthat, due to the spatial encoding dimension of the invention, even muchlarger numbers can be contemplated. For example, assaying 10,000 targetsper location×10,000 locations would generate 10⁸ different assays, andeven larger numbers than these can easily be contemplated, particularlyif spatial locations with resolution on the order of that of singlecells are utilized. Further, in embodiments where high-throughputdigital sequencing is employed, the sequences of at least 1,000 encodingprobes are typically determined in parallel. More typically, using adigital readout, it is desirable to obtain multiple sequence reads foreach assay (defined by a probe and a spatial location code). It isdesirable to obtain an average of at least 3 copies per assay, and moretypically at least 10 or at least 30 copies per assay, depending on thedesign of the experiment and requirements of the assay. For aquantitative readout with suitable dynamic range, it may be desirable toobtain at least 1,000 reads per assay. Therefore, if 1,000,000 assaysare carried out, the number of sequence reads may be 1 billion or more.With high-throughput digital sequencing, and allowing for redundancy,the sequence of at least 10,000 encoding probes are determined inparallel, or the sequence of at least 100,000, 500,000, 1,000,000,10,000,000, 100,000,000, 1,000,000,000 or more encoding probes aredetermined in parallel.

Assays

The assay portion of the assay systems of the present invention comprisethe following general steps: delivering probes and encoding agents wherethe encoding agents (in some embodiments pre-coupled to the probes) aredelivered to the sample according to a known spatial pattern, allowingthe probes to interact or react with biological targets in the sample,and, if the probes and encoding agents have not been pre-coupled,coupling the encoding agents to probes.

The samples of the present invention include virtually any biologicalsample or samples that can be affixed to a support or providedessentially in a two-dimensional manner, where the ability to tie anassayed biological target or activity back to the location within thebiological sample is important. Exemplary biological samples includetissue sections (e.g., including whole animal sectioning and tissuebiopsies), cell populations on slides or culture dishes, and the like.The assay systems of the invention are particularly advantageous in thatthey are compatible with numerous biological sample types, includingfresh samples, such as primary tissue sections, and preserved samplesincluding but not limited to frozen samples and paraformalin-fixed,paraffin-embedded (FFPE) samples. An important aspect of the assaysystems of the invention is that the biological samples are immobilizedon a substrate surface having discrete, independently measurable areas.

The biological targets to be detected can be any biological moleculesincluding but not limited to proteins, nucleic acids, lipids,carbohydrates, ions, or multicomponent complexes containing any of theabove. Examples of subcellular targets include organelles, e.g.,mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts,endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.

In some particular embodiments, the assay system is used to analyzenucleic acids, e.g., by genotyping, quantitation of DNA copy number orRNA transcripts, localization of particular transcripts within samples,and the like. FIG. 3 illustrates an overall scheme for an exemplaryassay for, e.g., detecting single nucleotide polymorphisms (SNPs) thatcan be used with the assay system of the invention. In FIG. 3, twooligonucleotide probes are provided. Each oligonucleotide probecomprises a target-specific region (located on either side of the SNP tobe analyzed) seen at 305 and 307, and ligation regions, seen at 301 and303, The oligonucleotide probes are allowed to hybridize to a targetnucleic acid (not shown) in the biological sample. At step 302, one ofthe oligonucleotide probes is extended to incorporate the SNP sequenceand ligated to the other probe to form an extended probe comprisingtarget nucleic acid region 309 and ligation regions 301 and 303.

Two encoding agents, both comprising a coding tag (seen at 315 and 317),a ligation region (seen at 311 and 313), and a primer region (seen at319 and 321) are combined with and ligated to the extended probe at step304 to form an encoded target-specific oligonucleotide. Again, incontrast with FIG. 1, the probes and encoding agents are delivered atseparate steps. Doing so allows use of the combinatorial embodimentsdescribed infra. In preferred embodiments, the encoding oligonucleotideswithin a pair of encoding oligonucleotides ligate specifically to oneside of the target sequence or the other (i.e., 5′ or 3′ of the targetsequence) in step 306. Also, typically, the ligation and primer regionsof the encoding oligonucleotides and probes are universal; that is, theset of ligation and primer regions used in constructing the probes andencoding oligonucleotides are constant, and only the target-specificregions of the probes and the coding tags of the encodingoligonucleotides differ. However, again in alternative embodiments, theligation and primer regions are not universal and differ between probesand encoding agents.

Following ligation, the encoded probes are eluted, pooled, and,optionally, sequencing adapters are added to the encoded probes via PCR.In alternative embodiments, sequencing primers may be ligated to theencoding oligonucleotides, or sequencing primer sequences can beincluded as part of the encoding oligonucleotide. As seen in FIG. 3,each sequencing adapter comprises primer region 319 or 321, compatiblewith the primer regions 319 and 321 on the encoded probes. The finalconstruct comprising first adapter 327, first primer region 319, firstcoding tag 315, ligation regions 311 and 301, target region 309,ligation regions 313 and 303, second coding tag 317, second primerregion 325 and second adapter 329 is now ready for input into a digitalhigh-throughput sequencing process.

A combination of extension and ligation reactions are exemplified inFIG. 3, but it should be appreciated that a variety of reactions may beused to couple the encoding oligonucleotides to the target-specificoligonucleotides, including ligation only (e.g., for oligonucleotidesthat hybridize to contiguous portions of the target nucleic acidsequence). Alternatively, an assay utilizing an additionaloligonucleotide, such as in the GOLDENGATE® assay (see Fan, et al., ColdSpring Symp. Quant. Biol., 68:69-78 (2003); (Ilumina, Inc., San Diego,Calif.)), may be employed.

In other embodiments, the assay system of the invention also can be usedto analyze peptides or proteins, the presence of antibodies, enzymaticand other protein activities, posttranslational modifications, activeand non-active forms of peptides, as well as peptide isoforms in abiological sample. Accordingly, the probes may comprise an active regionof an enzyme, a binding domain of an immunoglobulin, defined domains ofproteins, whole proteins, synthetic peptides, peptides with introducedmutations, aptamers and the like.

In certain aspects, the probes are substrates for enzymes or proenzymes,e.g., kinases, phosphatases, zymogens, proteases, or fragments thereof.In certain aspects, the probes are phosphorylation substrates used todetect proteins involved in one or more signal transduction pathways,e.g., a kinase or a phosphatase. In another specific aspect of theinvention, the probes are specific protease substrates that associateonly with individual proteases or classes of proteases. In otheraspects, the probes are different processed forms, isoforms and/ordomains of an enzyme. Protein-based probes are typically conjugated orotherwise linked to oligonucleotide encoding agents. The oligonucleotideencoding agents in this case would also include a nucleotide sequencecomponent that allows for identification of the protein probe.

In certain aspects, the present invention provides assays for evaluatingdifferences in the amount and/or activity of biological targets betweendifferent locations in a sample and/or between samples. The methodincludes determining a plurality of encoded results from the biologicalsample and evaluating the differences in quantity of the biologicaltargets at each location in the biological sample.

Combinatorial Embodiments

To maximize the efficiency of encoding, a combinatorial approach usingpairs of coding tags in the encoding oligonucleotides can be used. Byde-coupling the target-specific information and the coding tags, thenumber of oligonucleotides required is dramatically reduced, with aconcomitant decrease in cost.

FIG. 4 illustrates a general mechanism for one embodiment of acombinatorial encoding scheme of the assay systems of the invention,where nucleic acids in a representative tissue section (shown at 416)are assayed. FIG. 4 at A shows two target-specific/encodingoligonucleotide constructs 420 and 422 (e.g., formed between steps 302and 304 of FIG. 3) specifically bound to a target nucleic acid 402 ofinterest, The first encoded probe 420 comprises coding tag 408,associated with, e.g., a universal priming site for amplification of theassay products or an adapter to enable identification of the codingidentifiers using sequencing technologies 404. The second encoded probe422 comprises coding tag 406, associated with, e.g., a universal primingsite for amplification of the assay products or an adapter to enableidentification of the coding identifiers using sequencing technologies410.

FIG. 4 at B shows the spatial pattern that may be used for twentydifferent coding tags, a1 through a10 (coding tag 406 on encoded probe420) and b1 through b10 (coding tag 408 encoded probe 422). Coding taga1., for example, is deposited on the biological sample in ten discreteareas or spots (shown as the first horizontal line of spots in 412).Coding tag a2 is deposited on the biological sample in ten spots on thesecond horizontal line in 412. Coding tag a3 is deposited on thebiological sample in ten spots on the third horizontal line in 412, andso on. Whereas the “a” tags are deposited in ten horizontal rows, the“b” tags are deposited in ten vertical rows as shown in 414. Forexample, coding tag b1 is deposited on the biological sample in tendiscrete spots in the first vertical row of 414, coding tag b2 isdeposited on the biological sample in ten discrete spots in the secondvertical row of 414, and so on. Using such a configuration allows fortwenty coding tags to uniquely define 100 different locations on thebiological sample.

FIG. 4 at C shows a representative tissue section 416 coincident withcoding tag grid 418. The arrows show how the “a” coding tags and the “b”coding tags are deposited on grid 418 that is coincident with tissuesection 416. If, once sequenced, coding tags a1 and b4, e.g., areassociated with a target nucleic acid sequence, then that target nucleicacid sequence (i.e., biological target) was present in the tissuesection at location a1, b4.

FIG. 5 provides a simplified, specific example of the encoding scheme ofthe assay systems of the invention. FIG. 5 shows encodingoligonucleotides 510, comprising a1, a2, a3, a4 and b1, b3, b3 and b4.Target-specific oligonucleotides (TSOs) (probes) 1 and 2 are shown at520. A deposit or dispensing scheme is shown at 530. Like the gridexemplified in FIG. 4, encoding oligonucleotides a1 through a4 aredeposited in spots in a pattern (here, in a vertical pattern), andencoding oligonucleotides b1 through b4 are deposited in spots in apattern (here, a horizontal pattern). The grid though shown as a squarewith spots is actually a deposition pattern on a biological sample (notshown) such as tissue section 416 shown in FIG. 4.

The target-specific oligonucleotides are delivered to the biologicalsample, where the target-specific oligonucleotides hybridize to targetnucleic acids in the biological sample if target nucleic acids arepresent. Unhybridized target-specific oligonucleotides are then removed,e.g., by washing. The encoding oligonucleotides are then delivered tothe biological sample according to the spatial pattern shown at 530. Theencoding oligonucleotides are ligated (or, e.g., extended and ligated)to any target-specific oligonucleotides that hybridized to the targetnucleic acid in the biological sample, the ligated constructs are theneluted from the biological sample, pooled, and sequencing adapters areadded through, e.g., PCR or ligation, if the sequences were notpreviously included in the encoding oligonucleotides. The ligatedconstructs are sequenced by, e.g., high throughput or “next generation”sequencing.

The pool of resulting sequences is shown at 540. A sequence readout wasobtained for target-specific oligonucleotide 1 only at a4b1, a4b2, a1b3,a2b3, a3b3, a4b3 and a4b4 (positions shown with horizontal lines). Asequence readout was obtained for target-specific oligonucleotide 2 onlyat a1b1 (position shown with vertical lines). A sequence readout wasobtained for both target-specific oligonucleotides 1 and 2 at positionsa2b1, a3b1, a1b2, a2b2, and a3b2 (positions shown with cross-hatching).No sequence readout was obtained for either target-specificoligonucleotides at a1b4, a2b4 or a3b4 (positions shown withoutshading). Thus, in the biological sample on which the assay took placethe first target nucleic acid was detected in a large portion of theleft side and at the bottom of the biological sample, the second targetnucleic acid was detected only in the upper left portion of thebiological sample, and neither target nucleic acid was detected in theupper right portion of the biological sample. The differentialexpression of the two target nucleic acids now can be mapped back to thebiological sample and to the biological structures or cell types inthese locations in the biological sample, as shown in 550.

In addition to location information, information relating to relativeabundance of the encoded tags can be obtained. For example, if it isfound that there are ten times as many a4T1b1 sequences occurring in thedata set as compared to a4T1b2 sequences, this would indicate thattarget nucleic acid sequence 1 is ten times more abundant at the a4T1b1location than at the a4T1b2 location.

In the case of nucleotide analysis as shown in FIG. 3, by ligating thecoding tags directly to target-specific oligonucleotides, only 2ntarget-specific oligonucleotides are needed for n targets. For example,using the combinatorial approach outlined in FIG. 2, assaying 100different targets at 10,000 spatial locations would require 2×100target-specific oligonucleotides and 2×100 encoding oligonucleotides.The total count of assay oligonucleotides would be only 400 (200target-specific and 200 encoding), not counting universal primers. Incontrast, if the coding oligonucleotides were not decoupled from thetarget-specific oligonucleotides, (n×X positional codes)+(n×Y positionalcodes) would be needed, or in the above example, 20,000oligonucleotides, not counting universal primer sequences. Moreover,though the embodiments shown in FIGS. 2-5 depict a combinatorial schemeusing two encoding agents (coding tags), three, four or more encodingagents and coding tags may be used, and attached to the probe or oneanother by varying means and in varying combinations of steps.

Due to the spatial encoding aspect of the assay system of the invention,a large amount of information can be generated with even a modest numberof assays. For example, five or more biological targets assayed at fiveor more positions in the sample generates 25 or more combinations. Usingdigital sequencing as a readout, the optimum number of sequence readsper combination depends on the sensitivity and dynamic range required,and can be adjusted, For example, if for each combination on average 100reads are sampled, the total for 25 combination is 25,000 reads. If1,000 targets are assayed at 1,000 locations with an average samplingdepth of 1,000, then 10⁹ reads are required. These numbers, althoughlarge, are within the capacity of intrinsically parallel digitalsequencing methods, which can generate datasets of billions or eventrillions of reads in a reasonable timeframe and at a very low cost perread. Therefore, by varying the numbers of positions interrogated orbiological targets assayed, or both, and using digital sequencing, largeamounts of information can be obtained. In specific aspects, multiplelocations are interrogated for two or more biological molecules.

Reagent Delivery Systems

The reagent delivery system of the invention includes instrumentationthat allows the delivery of reagents to discrete portions of thebiological sample, maintaining the integrity of the spatial patterns ofthe encoding scheme. Reagent delivery systems of the assay systems ofthe invention comprise optional imaging means, reagent delivery hardwareand control software. Reagent delivery can be achieved in a number ofdifferent ways. It should be noted that reagent delivery may be to manydifferent biological samples at one time. A single tissue section hasbeen exemplified herein; however, multiple biological samples may beaffixed and analyzed simultaneously. For example, pions of a tissuesample can be analyzed in parallel and the data combined to build a 3Dmap.

Integral to the assay system of the invention is instrumentation thatallows for spatial patterning of reagents onto the biological sample.Technologies for formulating and delivering both biological molecules(e.g. oligonucleotides or antibodies) and chemical reagents (e.g., smallmolecules or dNTPs) are known in the art, and uses of these instrumentsystems are known to one skilled in the art and easily adaptable to theassay systems of the invention. One example of a suitable reagentdelivery system is the Labcyte™ Echo acoustic liquid handier, which canbe used to deliver nanoliter scale droplets containing biologicalmolecules with high precision and reproducibility. One skilled in theart could incorporate this reagent delivery device into the overallsystem, using software to specify the locations to which reagents shouldbe delivered.

Other instruments that can be used for the deposition of agents and/orcoding identifiers onto biological samples include, but are not limitedto, ink jet spotting; mechanical spotting by means of pin, pen orcapillary; micro contact printing; photochemical or photolithographicmethods; and the like. For several applications, it may be preferred tosegment or sequester certain areas of the biological samples into one ormore assay areas for different reagent distributions and/or biologicaltarget determination. The assay areas may be physically separated usingbarriers or channels.

In one exemplary aspect, the reagent delivery system may be a flow-basedsystem. The flow-based systems for reagent delivery in the presentinvention can include instrumentation such as one or more pumps, valves,fluid reservoirs, channels, and/or reagent storage cells. Reagentdelivery systems are configured to move fluid to contact a discretesection of the biological sample. Movement of the reagents can be drivenby a pump disposed, for example, downstream of the fluid reagents. Thepump can drive each fluid reagent to (and past) the reactioncompartment. Alternatively, reagents may be driven through the fluid bygravity. US Pub. Nos. 20070166725 and 20050239192 disclose certaingeneral-purpose fluidics tools that can be used with the assay systemsof the invention, allowing for the precise manipulation of gases,liquids and solids to accomplish very complex analytical manipulationswith relatively simple hardware.

In a more specific example, one or more flow-cells can be attached tothe substrate-affixed biological sample from above. The flow-cell caninclude inlet and outlet tubes connected thereto and optionally anexternal pump is used to deliver reagents to the flow-cell and acrossthe biological sample. The flow cells are configured to deliver reagentsonly to certain portions of the biological sample, restricting theamount and type of reagent delivered to any specific section of thebiological sample.

In another aspect, a microfluidic system can be integrated into thesubstrate upon which the biological sample is disposed or externallyattached on top of the substrate. Microfluidic passages for holding andcarrying fluid may be formed on and/or above the planar substrate by afluidics layer abutted to the substrate. Fluid reagents can be selectedand delivered according to selective opening and closing of valvesdisposed between reagent reservoirs.

Pumps generally include any mechanism for moving fluid and/or reagentsdisposed in fluid. In some examples, the pump can be configured to movefluid and/or reagents through passages with small volumes (i.e.,microfluidic structures). The pump can operate mechanically by exertinga positive or negative pressure on fluid and/or on a structure carryingfluid, electrically by appropriate application of an electric field(s),or both, among other means. Exemplary mechanical pumps may includesyringe pumps, peristaltic pumps, rotary pumps, pressurized gas,pipettors, etc. Mechanical pumps may be micromachined, molded, etc.Exemplary electrical pumps may include electrodes and may operate byelectrophoresis, electroendoosmosis, electrocapillarity,dielectrophoresis (including traveling wave forms thereof), and/or thelike.

Valves generally include any mechanism for regulating the passage offluid through a channel. Valves can include, for example, deformablemembers that can be selectively deformed to partially or completelyclose a channel, a movable projection that can be selectively extendedinto a channel to partially or completely block a channel, anelectrocapillary structure, and/or the like.

An open gasket can be attached to the top of the biological sample andthe sample and reagents can be injected into the gasket. Suitable gasketmaterials include, but are not limited to, neoprene, nitrile, andsilicone rubber. Alternatively, a watertight reaction chamber may beformed by a gasket sandwiched between the biological sample on thesubstrate and a chemically inert, water resistant material such as, butnot limited to, black-anodized aluminum, thermoplastics (e.g.,polystyrene, polycarbonate, etc), glass, etc.

In an optional embodiment, the assay system comprises imaging means todetermine features and organization of the biological sample ofinterest. The images obtained, e.g., may be used to design thedeposition pattern of the reagents, Imaging means are optional, as anindividual can instead view the biological sample using, e.g., amicroscope, analyze the organization of the biological sample, andspecify a spatial pattern for delivery assay reagents. If included, thedelivery system can comprise a microcircuit arrangement including animager, such as a CCD or IGFET-based (e.g., CMOS-based) imager and anultrasonic sprayer for reagent delivery such as described in US Pub. No.20090197326, which is incorporated herein by reference. Also, it shouldbe noted that although FIGS. 4 and 5 illustrate using a x,y gridconfiguration, other configurations can be used, such as, e.g.,following the topology of a tissue sample; targeting certain groups ofcells, cell layers and/or cell types in a tissue, and the like.

In yet another alternative, the reagent delivery system controls thedelivery of reagents to specific patterns on a biological sample surfaceusing semiconductor techniques such as masking and spraying. Specificareas of a biological sample can be protected from exposure to reagentsthrough use of a mask to protect specific areas from exposure. Thereagents may be introduced to the biological sample using conventionaltechniques such as spraying or fluid flow. The use of masked deliveryresults in a patterned delivery scheme on the substrate surface.

In a preferred aspect of the invention, the reagent deliveryinstrumentation is based on inkjet printing technology. There are avariety of different ink-jetting mechanisms (e.g., thermal,piezoelectric) and compatibility has been shown with aqueous and organicink formulations. Sets of independently actuated nozzles can be used todeliver multiple reagents at the same time, and very high resolutionsare be achieved.

In order to target specific sites of interest, an informative image ofthe biological sample to be assayed may be used to assist in the reagentdelivery methods and associated encoding scheme. Sample regions of thebiological sample can be identified using image processing (e.g., imagesof cell types differentiated by immunohistochemistry or other stainingchemistries) integrated with other features of the assay system. In someaspects, software is used to automatically translate image informationinto a reagent delivery pattern. A mechanism to register and align veryprecisely the biological sample for reagent delivery is thus animportant component of the assay systems of the invention. Mechanismssuch as the use of fiducial markers on slides and/or other very accuratephysical positioning systems can be adapted to this purpose.

The invention preferably comprises a complete suite of software tailoredto the assay system. Optionally, oligonucleotide design software is usedto design the encoding nucleotides (and in embodiments where nucleicacids are assayed, the target-specific oligonucleotides) for thespecific assay to be run, and may be integrated as a part of the system.Also optionally, algorithms and software for reagent delivery and dataanalysis (i.e., sequence analysis) may be integrated to determine assayresults. Integrated data analysis is particularly useful, as the type ofdataset that is generated may be massive as a consequence of scale.Algorithms and software tools that are specifically designed foranalysis of the spatially-associated data generated by the assaysystems, including pattern-analysis software and visualization tools,enhance the value of the data generated by the assay systems.

In certain aspects, the assay system comprises processes for making andcarrying out the quality control of reagents, e.g., the integrity andsequence fidelity of oligonucleotide pools. In particular, reagents areformulated according to factors such as volatility, stability at keytemperatures, and chemical compatibility for compatibility with thereagent delivery instrumentation and may be analyzed by instrumentationintegrated within the assay system.

Sequencing

Numerous methods can be used to identify the coding tags and probesequences in the encoded probes of the assay systems of the invention.The coding tags can be detected using techniques such as massspectroscopy (e.g., Maldi-T of, LC-MS/MS), nuclear magnetic resonanceimaging, or, preferably, nucleic acid sequencing. Examples of techniquesfor decoding the coding tags of the present invention can be found, forexample, in US Pub. No. 20080220434, which is incorporated herein byreference. For example, the coding tags may be oligonucleotide mass tags(OMTs or massTags). Such tags are described, e.g., in US Pub. No.20090305237, which is incorporated by reference in its entirety. In yetanother alternative, the encoded probes can be amplified and hybridizedto a microarray. This would require separate amplification reactions tobe carried out, in which each amplification is specific to a particularspatial code or subset of codes, accomplished by using code-specificprimers. Each amplification would also incorporate a differentresolvable label (e.g. fluorophor). Following hybridization, therelative amounts of a particular target mapping to different spatiallocations in the sample can be determined by the relative abundances ofthe resolvable labels.

In one particularly preferred aspect, the resulting coding tagsaccording to the assay system are substrates for high-throughput,next-generation sequencing, and highly parallel next-generationsequencing methods are used to confirm the sequence of the coding tags,for example, with SOLiD™ technology (Life Technologies, Inc.) or GenomeAnanlyzer (Illumina, Inc.). Such next-generation sequencing methods canbe carried out, for example, using a one pass sequencing method or usingpaired-end sequencing. Next generation sequencing methods include, butare not limited to, hybridization-based methods, such as disclosed ine.g., Drmanac, U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; andDrmanac et al, U.S. patent publication 2005/0191656;sequencing-by-synthesis methods, e.g., U.S. Pat. Nos. 6,210,891;6,828,100; 6,969,488; 6,897,023; 6,833,246; 6,911,345; 6,787,308;7,297,518; 7,462,449 and 7,501,245; US Publication Application Nos.20110059436; 20040106110; 20030064398; and 20030022207; Ronaghi, et al,Science, 281: 363-365 (1998); and Li, et al, Proc. Natl. Acad. Sci.,100: 414-419 (2003); ligation-based methods, e.g., U.S. Pat. Nos.5,912,148 and 6,130,073; and U.S. Pat. Appln Nos. 20100105052,20070207482 and 20090018024; nanopore sequencing e.g., U.S. Pat. ApplnNos. 20070036511; 20080032301; 20080128627; 20090082212; and Soni andMeller, Clin Chem 53: 1996-2001 (2007)), as well as other methods, e.g.,U.S. Pat. Appln Nos. 20110033854; 20090264299; 20090155781; and20090005252; also, see, McKernan, et al., Genome Res., 19:1527-41 (2009)and Bentley, et al., Nature 456:53-59 (2008), all of which areincorporated herein in their entirety for all purposes.

Applications of Assay System

It will be apparent to one skilled in the art upon reading the presentdisclosure that there are numerous important areas of biologicalresearch, diagnostics, and drug development that will benefit from ahigh throughput multiplexed assay system that can measure simultaneouslythe amount and spatial location of a biological target in a biologicalsample. For example, combining the ability to estimate the relativeabundance of different RNA transcripts with the ability to reconstructan image of spatial patterns of abundance across many locations, whichmay be as small as or even smaller than individual cells, in a tissueenables many different areas of basic research. The following areexemplary uses and are by no means meant to be limiting in scope.

In one example, 3-dimensional patterns of gene expression are determinedby analyzing a series of tissue sections, in a manner analogous to imagereconstruction in CT scanning, Such a method can be used to measurechanges in gene expression in disease pathology, e.g., in canceroustissue and/or a tissue upon injury, inflammation or infection. With theassay systems of the invention, more detailed information on geneexpression and protein localization in complex tissues is obtained,leading to new insights into the function and regulation both in normaland diseased states, and provides new hypotheses that can be tested. Forexample, an assay system of the invention may enable some of theinsights gained from many individual studies and larger programs likeENCODE (Birney, et al., Nature, 447:799-816 (2007)) and modENCODE to beintegrated at the tissue level. The assay systems also aid computationalefforts to model interacting networks of gene expression in the field ofsystems biology.

The assay systems also provide a novel approach to analysis of somaticvariation, e.g., somatic mutations in cancer or variability in responseto infectious organisms. For example, tumors are typically highlyheterogeneous, containing cancer cells as well as genetically normalcells in an abnormal local environment. Cancer cells undergo mutationand selection, and in this process it is not unusual for local clones todevelop. Identifying relatively rare somatic mutations in the context oftumors may enable the study of the role of key mutations in theselection of clonal variants. Transcriptional patterns associated withangiogenesis, inflammation, or other cancer-related processes in bothcancer and genetically normal cells can be analyzed for insights intocancer biology and assist in the development of new therapeutic agentsfor the treatment of cancers. In another example, individuals havevarying susceptibility to infectious organisms, and the assay systems ofthe invention can be used to study the interaction between microbes andtissues or the various cell types within the tissue.

Importantly, in addition to providing spatially-associated information,the invention allows a great increase in the sensitivity of detectingrare mutations, as signal to noise can he dramatically increased sinceonly a small location is assayed in any given reaction. In a typicalassay for rare mutations in a mixed sample, the sample is treated inbulk, i.e., nucleic acids are extracted from many cells into a singlepool. Thus, if a mutation is present in one cell in 10,000, it must bedetected against a background of normal DNA from ˜10,000 cells. Incontrast, with the assay systems of the invention many cells can beanalyzed, but individual cells or small groups of cells would beidentified by the spatial coding system. Therefore, in the assay systemsof the present invention, background is reduced by orders of magnitude,greatly increasing sensitivity. Furthermore, the spatial organization ofmutant cells can be observed, which may be particularly important indetecting key mutations in tissue sections in cancer. Already molecularhistological analyses are yielding insights into cancer biology and mayhave potential for use in diagnostics. The technology of the inventionpromises to greatly increase the power of such approaches.

The present invention provides assays, assay systems, and methods ofusing such assays in spatially encoded biological assays. The inventionprovides an assay system comprising one or more agents provided indefined spatial patterns on a substrate surface, and a detection systemfor identifying the presence or absence, relative amount, and locationof a biological molecule. Such biological molecules include, but are notlimited to, nucleic acids, peptides, carbohydrates, cellular components,and the like. The assay system is a novel multiplexing approach, as itallows multiple molecules and their respective multiple locations to beidentified in a single system using a unique encoding scheme. Thisencoding scheme uses both molecule-specific binding agents and codingidentifiers to provide a practical and cost-effective determination ofinformation on multiple biological molecules, including specificpositional information of such molecules in a biological sample, e.g., atissue section. The single molecule detection analysis using theencoding system also allows relative amounts of biological molecules tobe detected, thus providing information on expression levels,sequestering in specific locales, and the like.

The assay systems detect the presence or absence, and relative amount,of a biological molecule at more than one spatial location in a sample.In addition, the assays provide methods for doing this for multiplebiological molecules simultaneously. The assay systems utilize one ormore binding agents that specifically bind to the biological molecule ofinterest and unique coding identifiers associated with specific bindingagents. The detection system utilizes a method for identifying thepresence and spatial address of the agent binding based on the positiveand/or negative results that are obtained using detection of the agentand identifier and the encoding scheme of the spatial patterns on thesubstrate surface. In a specific aspect, the encoding scheme employslimited reagent delivery to the spatial patterns on the substratesurface, and access of the coding identifiers and/or binding agents toportions of the sample is controlled through such limited delivery.

In one aspect, the assay system detects the presence or absence andspatial location of a biological molecule based on the positive and/ornegative results that are obtained using limited reagent delivery andthe encoding scheme of the spatial patterns on the substrate surface.

The assay system and methods of the invention are based on relational,solid-state substrates with positions that represent specific spatiallocations within a biological sample, e.g., a cell, organelle or tissue.The ability to use encoding features to represent locations allowshigh-throughput analysis of the presence or absence, and relativeamount, of a biological molecule at more than one spatial location in asample. The encoding features also allow provide assaying of multiplebiological molecules at these multiple locations simultaneously.

A primary feature of the invention is the preservation of the spatialorganization of elements in a sample of interest through the use of anencoding scheme. For example, the assay may be designed to preserve therelative position of cells in a tissue, and the assay may interrogatethe individual cells for genomic DNA variation (including epigeneticmodifications), and RNA and protein expression.

In one specific aspect, the encoding scheme of the assay systemcomprises the use of two or more coding patterns, each comprisingregions defined by spatial patterns on the substrate surface. Forexample, the assay system can utilize an encoding scheme that comprisesa 2-dimensional grid format based on the discrete positioning of thebinding agents in the substrate surfaces. In another example, thespatial patterns may be based on more randomized cell locations, e.g.,the patterns on the substrate surface follow an underlying biologicalstructure rather than a strict, x,y grid pattern. This aspect includessystems with two or more substantially identical spatial patterns usingdifferent binding agents and/or coding identifiers, as well as systemshaving different patterns for different agents and/or codingidentifiers. The encoding scheme of the systems can be controlled bydelivery of different reagents to discrete regions on the substratesurfaces, which allows different reactions to take place onsubstantially similar agents of known location on the substratesurfaces.

In one specific aspect, the invention provides high resolution,high-throughput analysis of nucleic acids and/or expression levels thatprovides both detection and spatial identification of large numbers ofnucleic acids, e.g., DNA or RNA.

In another specific aspect, the invention provides high resolution,high-throughput analysis of proteins that provides both detection andspatial identification of large numbers of such proteins, e.g., kinasesor proteases.

Numerous reagent delivery systems can be used with the assay system ofthe invention. The primary criteria of such reagent delivery systems isthe ability to direct delivery of specific agents based on spatialpatterns on the substrate surface.

In one preferred aspect, the encoding scheme utilizes a reagent deliverysystem based on printing and informatics technologies to implement thespatial patterns used for identification and localization of thebiological materials. For example, the patterns found in the encodingscheme may be created using ink jet printing technology to providereagents at specific locations on one or more substrate surfaces. Thedesired patterns are set out in specific coding patterns on thesubstrate surface.

In certain aspects of the invention, the binding agents are immobilizeddirectly to the substrate surface, and the location of the bindingagents is known or determined prior to use of the substrate surface inthe assay system. In another aspect, the binding agents are immobilizedonto beads or other separate structural elements that are then providedin known locations on the substrate surface. In yet another aspect, thebinding agents may be provided in or on features of the substratesurface, e.g., provided in wells or channels.

In specific aspects of the invention, the binding agents are nucleicacids immobilized directly or indirectly to the substrate surface, e.g.,directly through the use of amino groups on the substrate surface orindirectly through the use of a linker. The location of the nucleic acidsequences is known or determined prior to use of the substrate surfacein the assay system. In another specific aspect, the nucleic acids maybe immobilized directly or indirectly onto beads that are then providedin known locations on the substrate surface. In yet another aspect, thenucleic acids may be provided in or on features of the substratesurface, e.g., provided in wells.

In these aspects involving nucleic acid agents, any methods of sequencedetermination can be used, e.g., sequencing, hybridization and the like.In a preferred aspect, nucleic acid sequencing, and preferablynext-generation sequencing, is used to decode the spatial encodingscheme in the assay system of the invention. This provides a very widedynamic range for very large numbers of assays, allowing for efficientmultiplexing.

In some aspects, the assay utilizes two or more oligonucleotides, theoligonucleotides comprising a universal primer region and a region thatcorrelates specifically to a single spatial pattern within the spatialencoding scheme. In a specific aspect, the assay comprises two allelespecific oligonucleotides and one locus specific oligonucleotides. Theseoligonucleotides allow the identification of specific SNPs, indels ormutations within an allele. This is useful in the identification ofgenetic changes in somatic cells, genotyping of tissues, and the like.

In other specific aspects of the invention, the binding agents arepeptides. In one aspect, these peptides are associated directly orindirectly to known locations on a substrate surface, e.g., usingbinding protein pairs or through oligonucleotide linkers complementaryto oligonucleotides on the substrate surface. In another aspect, thebinding agents are peptides are immobilized directly or indirectly ontobeads or other separate structural elements that are then provided inknown locations on the substrate surface. In yet another aspect, thepeptides may be provided in or on features of the substrate surface,e.g., provided in wells.

In yet other specific aspects of the invention, the binding agents arechemical entities (e.g., small molecules) that are coded, e.g. usingsequence tags or mass spectroscopy tags as coding identifiers. In oneaspect, these chemical entities can be are immobilized directly to thesubstrate surface. In another aspect, the binding agents are immobilizedonto beads or other separate structural elements that are then providedin known locations on the substrate surface. In yet another aspect, thebinding agents may be provided in or on features of the substratesurface, e.g., provided in wells.

The assay system of the invention can utilize various detectionmechanisms, based on the molecules to be detected and the reagentsneeded for such detection system. Exemplary methods that can be usedwith the assay systems of the invention are described in more detailbelow.

The Invention in General

The assay system and methods of the invention are based on relationalmethods that allow extraction of data to detect the presence or absenceand relative amount of a biological molecule, and the location of thismolecule in a sample having a distinct structure, e.g., a tissue sectionor other biological structure with distinct locations of specificbiological molecules. The encoding scheme used in these systemscorresponds to the structural elements of the sample, and theinformation obtained using a two-dimensional coding system is indicativeof the spatial addresses of these molecules in a sample of interest.

Integral to the assay system of the invention is a method for spatialpatterning of reagents. Technologies for formulating and delivering bothbiological molecules (e.g. DNA or antibodies) and chemical reagents(e.g., small molecules or dNTPs) have already been demonstrated, and useof these systems will be available to one skilled in the art and easilyadaptable upon reading this specification.

The assay design of the invention provides an accurate and easilyscalable spatial encoding system. The ability to deliver reagents in aspatially defined pattern together with software, reagents and protocolscomprises a novel and highly innovative assay system for spatialanalysis of various biological molecules and activities. This allows theassays to measure numerous biological functions in a meaningful spatialenvironment, including functions such as gene expression and peptidelocalization. The systems provide the potential to open a new analyticalwindow into the complex spatial patterns of cellular function andregulation in biological systems.

The biological molecules to be detected can be any biological moleculessuch as proteins, nucleic acids, lipids, carbohydrates, ions, ormulticomponent complexes containing any of the above. Further examplesof subcellular objects include organelles, e.g., mitochondria, Golgiapparatus, endoplasmic reticulum, chloroplast, endocytic vesicle,exocytic vesicles, vacuole, lysosome, etc.

FIG. 4 illustrates such a target-specific assay system foridentification of nucleic acid sequences in a sample. In this system,two reagents 420, 422 that specifically bind to a biological molecule ofinterest are associated with coding identifiers 406, 408 that encode fora spatial location in the sample. These coding identifiers 406, 408 areoptionally associated with sites that assist in their identification inthe assay format, e.g., universal priming sites 404, 410 foramplification of the assay products or adapters to enable identificationof the coding identifiers and the binding agents using sequencingtechnologies. The sample that is tested, here shown as a tissue section416 is encoded using the combination of the patterns 412, 414 createdusing the separate coding identifiers 406, 408 which provide a twodimensional code 418 that shows the location of any positive detectionof the biological molecule 402 as well as quantifying the biologicalmolecule 402 at each location assayed in the tissue.

The assay systems of the invention are particularly advantageous in thatthey are compatible with numerous samples types, such as fresh samples,such as primary tissue sections, and preserved samples including but notlimited to frozen samples and paraformalin-fixed, paraffin-embedded(FFPE) samples. An important aspect of the assay systems of theinvention is that the binding agents are immobilized on a substratesurface in discrete, independently measurable areas. These discreteareas can be formed by spatially selective deposition of the bindingagents on the substrate surface. Numerous methods can be used for thedeposition of the agent and the coding identifiers associates with theagent. For example, the coding identifiers can be delivered together orseparately from the agent. If delivered together they can be attached(e.g., synthesized as a single molecule or attached through ligation ora chemical coupling mechanism) or simply mixed together to be attachedafter delivery to the substrate. In a preferred aspect, the agent andthe coding identifier are made separately, mixed together forattachment, and delivered either attached or as a mixture to be attachedon the surface. In a specific aspect the binding agents are deliveredgenerally over the substrate surface and the coding identifiers aredelivered in a pattern-specific manner.

Examples of methods that can be used for deposition of agents and/orcoding identifiers onto the substrate surface include, but are notlimited to, ink jet spotting, mechanical spotting by means of pin, penor capillary, micro contact printing, fluidically contacting themeasurement areas with the biological or biochemical or syntheticrecognition elements upon their supply in parallel or crossed microchannels, upon exposure to pressure differences or to electric orelectromagnetic potentials, and photochemical or photolithographicimmobilization methods.

For several applications, it may be preferred to arrange the substratesinto segments of one or more measurement areas for reagent distributionand agent determination. These regions may be physically separated usingbarriers or channels. They may still comprise several additionaldiscrete measurement areas with agents that are different or indifferent combination from each other.

In certain aspects, the present invention provides a method, e.g., amachine-based method, for evaluating changes in the presence and/orlocation of a biological molecule over time. The method includesproviding a plurality of encoded array results representative of thebiological molecule over time and evaluating the differences indetection and/or localization of the biological molecules.

Nucleic Acid Detection and Localization

In a particular aspect, the assay system is used to analyze nucleicacids, e.g genotyping, gene expression analysis, localization ofparticular transcripts within samples, and the like.

Genotyping may be performed using any technique known to those of skillin the art. Preferred techniques permit rapid, accurate determination ofmultiple variations with a minimum of sample handling. Some examples ofsuitable techniques involve but are not limited to direct DNAsequencing, capillary electrophoresis, hybridization, allele-specificprobes or primers, single-strand conformation polymorphism analysis,nucleic acid arrays, bead arrays, restriction fragment lengthpolymorphism analysis, cleavage fragment length polymorphism analysis,random amplified polymorphic DNA, ligase detection reaction,heteroduplex or fragment analysis, differential sequencing with massspectrometry, atomic force microscopy, pyrosequencing, FRET (e.g.,TaqMan (Applied Biosystems, Inc., Foster City, Calif.) and MolecularBeacon (Stratagene, La Jolla, Calif.) assays), and other relatedtechniques. Several methods for DNA sequencing are well known andgenerally available in the art. See, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,New York) (2001); Ausubel, et al., Current Protocols in MolecularBiology (John Wiley and Sons, New York) (1997), Twyman, et al. (2003)“Techniques Patents for SNP Genotyping”, Pharmacogenomics 4(1):67-79;and Kristensen, et al. (2001) “High-Throughput Methods for Detection ofGenetic Variation”, BioTechniques 30(2):318-332. For details on the useof nucleic acid arrays (DNA chips) for the detection of, for example,SNPs, see U.S. Pat. No. 6,300,063 issued to Lipshultz, et al., and U.S.Pat. No. 5,837,832 to Chee, et al., HuSNP Mapping Assay, reagent kit anduser manual, Affymetrix Part No. 90094 (Affymetrix, Santa Clara,Calif.). The molecular inversion probe (MIP) assay format (Hardenbol etal., 2003) is another example of a highly multiplexable assay that maybe used with the assay systems of the invention.

In one exemplary and preferred method for analyzing nucleic acids usingthe assay system of the invention, the detection of nucleic acids usestwo allele-specific oligonucleotides and a locus specificoligonucleotide. The assay methods are carried out according to thestrategy outlined in FIG. 2 using next-generation sequencing or anotherhighly parallel nucleic acid assay technology. In this assay, a set oftwo oligonucleotides is designed to hybridize to each target sequence,with a common oligonucleotide and two unique coding identifiers. Theallele can be determined, e,g, by primer extension of the locus specificoligonucleotide. Following primer extension and ligation, an amplifiabletemplate is formed with universal primer sequences at either end. Assayoligonucleotides are annealed to a template and enzymatic reactions areused to join the two oligonucleotides only when both are correctlyannealed. The detection techniques and read out parameters used in thissystem of the invention include a much shorter tag than theoligonucleotides used in the assays that are based on capture byhybridization. These shorter tags are designed to be read out bysequencing or, preferably, used to ligate codes onto both ends of thefragment as illustrated in FIG. 2.

In FIG. 3, two target-specific assay oligonucleotides are ligatedtogether 302 following in situ hybridization to target sequences. At thesame time, encoding oligonucleotides containing tag sequence sets X andY are ligated 304 to the target specific oligonucleotides.Oligonucleotides containing X ligate specifically to one side of thetargeting construct and oligonucleotides containing Y ligate to theother. The oligonucleotides contain universal primer sites P1 and P2.Following ligation, the constructs are eluted and, optionally,sequencing adapters can be attached 306, e.g., via PCR.

In one preferred aspect, the final construct created from the assaymethod is a substrate for next-generation sequencing, and highlyparallel next-generation sequencing methods are used to confirm thesequence of constructs. Such sequencing methods can be carried out, forexample, using a one pass sequencing method or using paired-endsequencing. Next generation sequencing methods include, but are notlimited to, hybridization-based methods, such as disclosed in Drmanac,U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al,U.S. patent publication 2005/0191656, and sequencing by synthesismethods, e.g., Nyren et al, U.S. Pat. No. 6,210,891; Ronaghi, U.S. Pat.No. 6,828,100; Ronaghi et al (1998), Science, 281: 363-365;Balasubramanian, U.S. Pat. No. 6,833,246; Quake, U.S. Pat. No.6,911,345; Li et al, Proc. Natl. Acad. Sci., 100: 414-419 (2003); Smithet al, PCT publication WO 2006/074351; use of reversible extensionterminators, e.g., Turner, U.S. Pat. No. 6,833,246 and Turner, U.S. Pat.No. 6,833,246 and ligation-based methods, e.g., Shendure et al (2005),Science, 309: 1728-1739, Macevicz, U.S. Pat. No. 6,306,597; whichreferences are incorporated by reference. Soddart et al., PNAS USA. 2009Apr. 20; Xiao et al., Nat Methods. 2009 March; 6(3):199-201. Epub 2009Feb. 8.

To maximize the efficiency of encoding, a combinatorial approach usingpairs of oligonucleotides can be used. For example, with only two setsof 100 codes, a substrate can theoretically encode up to 10,000locations. The number of assay oligonucleotides required is dramaticallyreduced, the cost decreased, and the robustness of the approachincreased by decoupling the coding sequences from the genome-specificsequences. Alternative assay formats can also be used (e.g. ligation orprimer extension followed by ligation).

By ligating the codes on separately, only 2n target-specific assayoligonucleotides are needed for n targets. For example, assaying 100different targets at 10,000 spatial locations would require 2×100targeting oligonucleotides and 2×100 encoding oligonucleotides, using acombinatorial approach outlined in FIG. 2. The total count of assayoligonucleotides would be only 400 (200 target-specific and 200encoding), not counting universal primers. In contrast, if the codingoligonucleotides were not decoupled, (n×X positional codes)+(n×Ypositional codes) would be needed, or in the above example, 20,000oligonucleotides, not counting universal primer sequences.

Due to the matrix system of the invention, a large amount of informationcan be obtained even using five or more positions interrogated for fiveor more biological molecules. By varying one or the other of these,large amounts of information can be obtained, both in terms of locationsand/or specific biological In specific aspects, multiple locations areinterrogated for two or more biological molecules. As an example, foreach datapoint ˜1,000 reads may be sampled, for a total of −10E9 readsfor 10E6 datapoints.

Peptide Detection Systems

The assay system of the invention can be used to analyze biologicalmolecules using peptide agents that are associated with the substratesurface in a spatial pattern. Such peptides may comprise an activeregion of an enzyme, a binding domain of an immunoglobulin, defineddomains of proteins, whole proteins, synthetic peptides, peptides withintroduced mutations, etc.

The assay system of the invention allows the identification and spatiallocation of various forms of peptides, including isoforms and peptidesthat have undergone posttranslational modification. Importantly, certainaspects of the invention allow the identification of active versusnon-active forms of such peptides in a sample. This allows theidentification of the presence or absence of specific peptide isoforms,and also acts as a proxy for identification of peptide activity in asample.

In certain aspects of the invention, the binding agents associated withthe substrate surfaces of the assay system include substrates forenzymes or proenzymes, e.g., a kinase, a phosphatase, a zymogen, aprotease, or a fragment thereof. In certain aspects, the binding agentsassociated with the substrate surfaces are phosphorylation substratesused to detect proteins involved with one or more signal transductionpathways, e.g., a kinase or a phosphatase. In another specific aspect ofthe invention, the binding agents are specific protease substrates thatassociate only with individual or classes of proteases. In otheraspects, the binding agents on the substrate surface are differentprocessed forms, isoforms and/or domains of an enzyme.

Reagent Delivery

The reagent delivery system of the invention can be any system thatallows the delivery of reagents to discrete portions of the array inorder to keep the integrity of the defined spatial patterns of theencoding scheme. Such discrete delivery can be achieved in a number ofdifferent ways.

In one exemplary aspect, the reagent delivery system can be a flow-basedsystem. The flow-based systems for reagent delivery in the presentinvention can include one or more pumps, valves, fluid reservoirs,channels, and/or reagent storage cells. Such a reagent delivery systemis configured to move fluid in contact with a discrete section of thesubstrate surface. Movement of the reagents can be driven through afluid by a pump disposed, for example, downstream of the fluid reagents.The pump can drive each fluid reagent to (and past) the reactioncompartment. Alternatively, the reagents may be driven through the fluidby gravity.

US Appln Nos. 20070166725 and 20050239192 disclose certaingeneral-purpose fluidics tools that can be used with the assay systemsof the invention. These allow the precise manipulation of gases, liquidsand solids to accomplish very complex analytical manipulations withrelatively simple hardware.

In a more specific example, one or more flow-cells can be attached tothe substrate from above. The flow-cell can include inlet and outlettubes connected thereto and optionally an external pump can be used todeliver the sample or reagents to the flow-cell and across thesubstrate. The flow cell is configured to deliver reagents only tocertain portions of the array, restricting the amount and type ofreagent delivered to any specific section of the array.

In another aspect, a microfluidic system can be integrated into thesubstrate or externally attached on top of the substrate. Microfluidicpassages for holding and carrying fluid can be formed on and/or abovethe planar substrate by a fluidics layer abutted to the substrate. Fluidreagents can be selected according to selective opening and closing ofvalves disposed between reagent reservoirs.

Pumps generally include any mechanism for moving fluid and/or reagentsdisposed in fluid. In some examples, the pump can be configured to movefluid and/or reagents through passages with small volumes (i.e.,microfluidic structures). The pump can operate mechanically by exertinga positive or negative pressure on fluid and/or on a structure carryingfluid, electrically by appropriate application of an electric field(s),or both, among others. Exemplary mechanical pumps may include syringepumps, peristaltic pumps, rotary pumps, pressurized gas, pipettors, etc.The mechanical pumps may be micromachined, molded, etc. Exemplaryelectrical pumps can include electrodes and may operate byelectrophoresis, electroendoosmosis, electrocapillarity,dielectrophoresis (including traveling wave forms thereof), and/or thelike.

Valves generally include any mechanism for regulating the passage offluid through a channel. The valves can include, for example, deformablemembers that can be selectively deformed to partially or completelyclose a channel, a movable projection that can be selectively extendedinto the channel to partially or completely block the channel, anelectrocapillary structure, and/or the like.

In yet another aspect, an open gasket can be attached to the top of thesubstrate and the sample and reagents can be injected into the gasket.Suitable gasket materials include, but are not limited to, neoprene,nitrile, and silicone rubber. Alternatively, a watertight reactionchamber formed by a gasket sandwiched between the substrate and achemically inert, water resistant material such as, but not limited to,black-anodized aluminum, thermoplastics (e.g., polystyrene,polycarbonate, etc), glass, etc.

In a specific aspect of the present invention, the delivery system cancompose a microcircuit arrangement including an imager, such as a CCD orIGFET-based (e.g., CMOS-based) imager and an ultrasonic sprayer forreagent delivery such as described in US Appln No. 20090197326, which isincorporated herein by reference.

In yet another aspect of the invention, the reagent delivery systemcontrols the delivery of reagents to specific patterns on a substratesurface using semiconductor techniques such as masking and spraying.Specific areas of a substrate surface can be protected from exposure toreagents through use of a mask to protect specific areas from exposure.The reagents may be introduced to the substrate using conventionaltechniques such as spraying or fluid flow. The use of the maskedsubstrate delivery results in a patterned delivery scheme on thesubstrate surface.

In a preferred aspect of the invention, the reagent deliveryinstrumentation is based on inkjet printing technology. There are avariety of different ink-jetting mechanisms (e.g., thermal,piezoelectric) and compatibility has been shown with aqueous and organicink formulations. Sets of independently actuated nozzles can be used todeliver multiple reagents at the same time, and very high resolutionscan be achieved.

Software for Use in the Assay System

In order to target specific sites of interest, an informative image ofthe biological section to be analyzed can be used to assist in thereagent delivery methods and associated encoding scheme. Sample regionscan be identified using image processing (e.g., images of cell typesdifferentiated by immunohistochemistry or other staining chemistries)integrated with the other features of the assay system. In some aspects,software is used to automatically translate this information into areagent delivery pattern. A mechanism to register and align veryprecisely the biological sample in a targeting system is thus apreferred component of the assay systems of the invention. Mechanismssuch as the use of fiducial markers on slides and other very accuratephysical positioning systems can be adapted to this purpose.

Additional software components will also be key components that will bepart of a complete assay system. The invention thus preferably comprisesa complete suite of software tailored to the assay system. Optionally,oligonucleotide design software will be customized for the specificassay to be run, and may be integrated as a part of the system. Alsooptionally, algorithms and software for data analysis may be integratedto assist in determination of results of the assays. This can beespecially useful, as the type of dataset that will be generated will benovel, particularly as a consequence of scale. The ability to providealgorithms and software tools that are specifically designed foranalysis of spatially-associated data for significant patterns,including pattern-analysis software and visualization tools, is a novelfeature that will enhance the value of the data generated by the assaysystems.

In certain aspects, the assay system will comprise processes for makingand carrying out quality control of reagents, e.g., the integrity andsequence fidelity of oligonucleotide pools. In particular, reagents willneed to be formulated for compatibility with the reagent deliveryinstrumentation. Factors such as volatility, stability at keytemperatures, and chemical compatibility can be optimized by thoseskilled in the art upon reading the present disclosure.

Applications of Assay System

It will be apparent to one skilled in the art upon reading the presentdisclosure that there are numerous very important areas of biologicalresearch, diagnostics, and drug development that will benefit from ahigh throughput means to simultaneously measure the presence or absenceand spatial location of a biological molecule in a sample. For example,this technology combining the ability to analyze semi-quantitatively theexpression of many different genes with the ability to image the spatialorganization of expression across many cells in a tissue is enabling formany different areas of basic research. The following are exemplary usesand are by no means meant to be limiting in scope.

In one example, 3-dimensional patterns of expression can be determinedby analyzing a series of tissue sections, in a manner analogous to imagereconstruction in CT scanning. This can be used to measure changes ingene expression in disease pathology, e.g., in cancerous tissue and/or atissue upon injury, inflammation or infection. With the assay systems ofthe invention, more detailed information on gene expression and proteinlocalization in complex tissues can be obtained. This may lead to newinsights into the function and regulation both in normal and diseasedstates, and is likely to provide new hypotheses that can be tested. Forexample, a system of the invention may enable some of the insightsgained from many individual studies and larger programs like ENCODE(Birney et al., 2007) and modENCODE to be integrated at the tissuelevel. The assay systems will also aid in computational efforts to modelinteracting networks of gene expression in the field of systems biology.

The assay systems also provide a novel approach that enables theanalysis of somatic variation, e.g., somatic mutations in cancer orvariability in response to infectious organisms. For example, tumors aretypically highly heterogeneous, containing cancer cells as well asgenetically normal cells in an abnormal local environment. Cancer cellsundergo mutation and selection, and in this process it is not unusualfor local clones to develop. Identifying relatively rare somaticmutations in the context of tumors may enable the study of the role ofkey mutations in the selection of clonal variants. Transcriptionalpatterns associated with angiogenesis, inflammation, or other cancerrelated processes in both cancer and genetically normal cells can beanalyzed for insights into cancer biology and assist in the developmentof new therapeutic agents for the treatment of cancers.

In another example, different people have varying susceptibility toinfectious organisms, and much of this may be to underlying geneticdifferences in individuals and/or populations. Identifying thesedifferences will aid in an understanding of the underlying diseasepathologies and assist in the development of vaccines or therapeutics toprevent or ameliorate these disease states.

Importantly, in addition to providing spatially associated information,the technology of the invention will allow a great increase in thesensitivity of detecting rare mutations. The reason is that signal tonoise can be dramatically increased because the approach of theinvention assays a small location in any given reaction. In a typicalassay for rare mutations in a mixed sample, the sample is treated inbulk, i.e. nucleic acids are extracted from many cells into a singlepool. Thus, if a mutation is present in 1 cell in 10,000, it must bedetected against a background of normal DNA from ˜10,000 cells. Incontrast, with the systems of the invention many cells can be analyzed,but individual cells or small groups of cells would be identified by thespatial coding system. Therefore, the background can be reduced byorders of magnitude, greatly increasing sensitivity. Furthermore, thespatial organization of mutant cells can be observed. This may beparticularly important in detecting key mutations in tissue sections incancer. Already, molecular histological analyses are yielding insightsinto cancer biology and may have potential for use in diagnostics (Choeet al., 2003). The technology of the invention promises to greatlyincrease the power of such approaches.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventor regards as his invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Example 1: Initial Proof of Concept of Encoding Scheme

As an initial proof of concept, a model system is developed using amicroarray to demonstrate a working single-plex assay. The basic designvalidates the concept of the assay, and establishes a working assayprior to addressing issues related to the analysis of a more complicatedbiological sample. Conventional sequencing is used as a readout for thisproof of concept.

A microarray is used as a proxy for a tissue section. The targetsequences of the microarray are fully specified, so that the compositionof the targets are known and can be varied systematically. Syntheticoligonucleotide templates are attached to a glass slide via a 5′ aminomodification. Each slide has a single oligonucleotide template sequence,and the assays that are carried out may employ either ligation, orextension followed by ligation as this may be useful in determiningcertain polymorphisms.

Once the in situ part of the assay is complete, the reaction productsare eluted and analyzed by qPCR to determined presence or absence of aproduct and estimate yield, and by conventional sequencing to determinethe structure of the assay products. The single plex assays that aretested include appropriate positive and negative controls, and a singlenucleotide variant (SNV) to check ability to discriminate single basevariants.

Example 2: Scalability

The complexity of the assay system is increased to establish scalabilityof the assay for use in high throughput studies. Scalability of both thespatial encoding and assay systems is demonstrated by carrying out a24-plex×24-site assay using a microarray model system.

The amount of biological target, here a DNA target sequence, at eachassay location is systematically varied on microarray substrate. Forexample, in a microarray with 50 micron spot size (center to center), a1 mm² area contains ˜400 spots. The region around each site isoptionally occupied by a region that is devoid of these spots to allowindividual resolvability of the target sequences. Alternatively, thespots may be clustered, with two or more directly adjacent spotssurrounded by or adjacent to a region that is devoid of targetsequences.

In order to demonstrate that spatial encoding is accurate, the sitescomprise different target compositions to show that the assay readoutmatches the expected composition of each site. With 24 target sequences,a simple digital pattern is made with each site having a different setof 12 targets present and 12 targets absent, to make a binary code(0=absent, 1=present). The assay readout is then determined to show thatthe detected regions match the expected signal after spatial decoding.In this particular example, the code space is large enough (2²⁴) so thateven a few errors would not result in different codes being mixed up.Moreover, this design allows identification of errors and allows anestimation not only of accuracy of spatial encoding but also of accuracycalling the presence or absence of target sequences.

In an exemplary aspect, a 4×4 arrangement of 16 sequences is used forthe array configuration. A white square indicates that the sequence isabsent and a black square that it is present, i.e. 8 of the 16 possiblesequences are present in this sample. In a different sample, a differentpattern of absent and present sequences can be constructed. In this way,unique patterns are associated with spatial locations so that theaccuracy of spatial encoding can be measured.

The ability to detect quantitative differences is evaluated bygenerating dose-response curves for each of the 24 assays that arecarried out at each site in a 24-site assay. This allows estimation ofthe limit of detection, dynamic range, and power to detect a givenfold-change across the range.

In one aspect, a latin square design is used to represent individualtargets at different ratios by varying the number of features for eachtarget. In other words, with multiple spots in a site, the number ofspots allocated to each of the 24 target sequences can be varied andeach of the 24 sites can have a different composition. A 1×3 inchmicroarray is sufficiently large to permit multiple replicates. Thislarger set of 24 sequences will require deconvolution, and this isaccomplished using high throughput techniques such as next-generationsequencing technologies (e.g., SOLiD™ technology (Life Technologies,Inc., Carlsbad, Calif.) or Genome Analyzer (Illumina, Inc., San Diego,Calif.)). The use of the 24-plex assay demonstrates both the accuracy ofspatial encoding and decoding, and the quantitative response of theassay system.

Example 3: Adaptation of the Assay to Preserved Samples

Genomic DNA is assayed as a proof of concept for assaying RNA, as itprovides a way to establish a single-copy reference signal. Once aworking assay is developed for FFPE samples, it is adapted to an RNAassay. To this end, assay oligonucleotide concentrations are assayed toensure compatibility with high multiplexing. Assuming a cell diameter of10 microns, and delivery of a 10 micron diameter reagent droplet to anindividual cell, the volume of the droplet will be ˜500 μl and cancontain ˜3×10¹¹ molecules at a 1 μM concentration. To assay 1,000 targetsequences in 10,000 cells, ˜2,000 targeting oligonucleotides would berequired in a droplet. Therefore, each droplet could contain ˜160million copies of each assay oligo, a vast excess over the few thousandtarget sequences in a cell.

The handling of small absolute numbers of product molecules generatedfrom very small or compromised samples are enhanced to counter the issueof low recovery efficiency; that is, elution is efficient and lossesresulting from adsorption of molecules to surfaces are prevented. Anapproach to addressing the latter issue is to include a carriermaterial, such as glycogen or carrier nucleic acids.

Example 4: Adapting the Assay to a Biological Sample

A control RNA template is immobilized to a solid support in order tocreate an artificial system. The assay is performed using T4 DNA ligase,which can repair nicks in DNA/RNA hybrids. Assays are carried out onmatched slides, or different sections of the same slide, where in onecase gDNA is assayed and in the other RNA is assayed. When assaying gDNAthe slide can be pretreated with RNase, and when assaying RNA the slideis pretreated with DNase. Results of the assay are confirmed byextracting gDNA or RNA and quantitating the relative amounts by qPCR orRT-qPCR respectively.

In order make the tissue section RNA assays as informative as possible,pre-existing information on expression levels in specific tissues totarget transcripts across a range of abundances are used in the assaydesign. Both high abundance transcripts, as well as some medium and lowabundance transcripts, are targeted to enable an initial assessment ofthe quantitative performance characteristics of the assay.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims. In the claims thatfollow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

I claim:
 1. A method of determining the presence or abundance of aplurality of biological molecules in a location of interest in abiological sample comprising: (a) contacting the biological sample witha first and a second probe for each of the plurality of biologicalmolecules, wherein a first and second probe for each biological moleculeeach comprise a sequence that is complementary to a sequence in thebiological molecule, and when the first and second probes are hybridizedto the biological molecule, the first and second probes are capable ofbeing ligated together; (b) imaging the biological sample to identifythe location of interest; (c) ligating first and second probes that arehybridized to a biological molecule to generate a ligation product(s)having a sequence; (d) separating the ligation product(s) from thelocation of interest in the biological sample; and (e) determining thesequence of the separated ligation product(s), thereby determining thepresence or abundance of the plurality of biological molecules at thelocation of interest in the biological sample, wherein step (b) isperformed before step (d).
 2. The method of claim 1, further comprising,after step (a), washing unhybridized first and second probes from thebiological sample.
 3. The method of claim 1, wherein step (b) isperformed before step (a), between step (a) and step (c), or betweenstep (c) and step (d).
 4. The method of claim 1, wherein the pluralityof biological molecules is RNA or DNA.
 5. The method of claim 1, whereinthe plurality of biological molecules represents a transcriptionalpattern in the location of interest of the biological sample.
 6. Themethod of claim 1, wherein the support is a slide or a culture dish. 7.The method of claim 1, wherein the biological sample is affixed to asupport.
 8. The method of claim 1, wherein the biological sample affixedto the support is a paraformalin-fixed, paraffin-embedded (FFPE) orfrozen sample.
 9. The method of claim 1, wherein the imaging isperformed using microscopy.
 10. The method of claim 1, determining thesequence of the separated ligation product(s) includes the performanceof high-throughput, next-generation sequencing.
 11. The method of claim1, further comprising a step of amplifying the ligation product(s)between the separating step and the determining step.
 12. A method ofdetermining the presence or abundance of a plurality of biologicalmolecules in a location of interest in a biological sample comprising:(a) imaging a biological sample to identify the location of interest;(b) contacting the location of interest in the biological sample with afirst and a second probe for each of the plurality of biologicalmolecules, wherein a first and second probe for each biological moleculeeach comprise a sequence that is complementary to a sequence in thebiological molecule, and when the first and second probes are hybridizedto the biological molecule, the first and second probes are capable ofbeing ligated together; (c) ligating first and second probes that arehybridized to a biological molecule in the location of interest in thebiological sample to generate a ligation product(s) having a sequence;(d) separating the ligation product(s) from the location of interest inthe biological sample; and (e) determining the sequence of the separatedligation product(s), thereby determining the presence or abundance ofthe plurality of biological molecules in the location of interest in thebiological sample.
 13. The method of claim 12, further comprising, afterstep (b), washing unhybridized first and second probes from thebiological sample.
 14. The method claim 12, wherein the plurality ofbiological molecules is RNA or DNA.
 15. The method of claim 14, whereinthe plurality of biological molecules represents a transcriptionalpattern in the location of interest of the biological sample.
 16. Themethod of claim 12, wherein the support is a slide or a culture dish.17. The method of claim 12, wherein the biological sample is affixed toa support.
 18. The method of claim 12, wherein the biological sampleaffixed to the support is a paraformalin-fixed, paraffin-embedded (FFPE)or frozen sample.
 19. The method of claim 12, wherein the imaging isperformed using microscopy.
 20. The method of claim 12, determining thesequence of the separated ligation product(s) includes the performanceof high-throughput, next-generation sequencing.
 21. The method of claim12, further comprising a step of amplifying the ligation product(s)between the separating step and the determining step.
 22. A method ofdetermining the presence or abundance of a plurality of biologicalmolecules in a location of interest in a biological sample comprising:(a) imaging a biological sample to identify the location of interest;(b) contacting the location of interest in the biological sample with afirst and a second probe for each of the plurality of biologicalmolecules, wherein a first and second probe for each biological moleculeeach comprise a sequence that is complementary to a sequence in thebiological molecule, and when the first and second probes are hybridizedto the biological molecule, the first and second probes are capable ofbeing ligated together; (c) ligating first and second probes that arehybridized to a biological molecule in the biological sample to generatea ligation product(s) having a sequence; (d) separating the ligationproduct(s) from the biological sample; (e) determining the sequence ofthe separated ligation product(s), thereby determining the presence orabundance of the plurality of biological molecules in the location ofinterest in the biological sample.
 23. The method of claim 22, furthercomprising, after step (b), washing unhybridized first and second probesfrom the biological sample.
 24. The method claim 22, wherein theplurality of biological molecules is RNA or DNA.
 25. The method of claim22, wherein the plurality of biological molecules represents atranscriptional pattern in the location of interest of the biologicalsample.
 26. The method of claim 22, wherein the support is a slide or aculture dish.
 27. The method of claim 22, wherein the biological sampleis a frozen sample or a paraformalin-fixed, paraffin-embedded (FFPE)sample.
 28. The method of claim 22, determining the sequence of theseparated ligation product(s) includes the performance ofhigh-throughput, next-generation sequencing.
 29. The method of claim 22,further comprising a step of amplifying the ligation product(s) betweenthe separating step and the determining step.